The rapid growth of nanotechnology (reported from the Congressional Research Service on August 29, 2013 projected a $3.1 trillion nanotechnology product revenue by 2015), high-cost in animal research, and increasing emphasis on animal well-being present a grand challenge and urgent need to develop viable in vitro means to understand the health risks of nanomaterials. Our long-term goal is to develop reliable in vitro methods to access the health effects of manufactured nanomaterials (NMs), and with this knowledge, to improve the materials' biocompatibility, and develop therapeutic strategies for disease prevention and intervention. This R21 plan proposes a completely new approach for investigation of nanomaterials-cell interactions that are of in vivo relevancy. A microfluidic delivery system will be integrated with the existing instrument of combined laser scanning confocal microscope and atomic force microscope (AFM) in the PI's laboratory. The microfluidic system enables delivery of nanomaterials with designed functionality and quantity to the designated sites of living cells, such as near membrane and intracellularly. The responses of cells will be monitored in situ using high resolution AFM imaging and single cell mechanics (PI team, US patent, 2012), from which significant responses will be identified. The nanomaterials treatments will then be replicated and monitored by confocal imaging to further reveal the nanoparticle-cell interactions intracellularly. The outcomes shall deepen our understanding of nanomaterials-cell interactions and demonstrate the concept that better in vivo mimetics may be achieved by eliminating the changes in NMs' states due to the delivery pathways such as interactions with culture media. Upon completion of this R21 research, we envision that cell mechanics (as a readout of cellular response) in conjunction with accurate delivery could be automated using Microelectromechanical systems (MEMs) platform, which has sufficient simplicity and throughput for R&D activities. Overall, this R21 shall provide a platform technology to enable important advances to reach the eventual goal of understanding and predicting nanotoxicity without using animal models.